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Proteomics Reveals Ablation of Placental Growth Factor Inhibits the Insulin Resistance

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Proteomics Reveals Ablation of Placental Growth Factor Inhibits the Insulin Resistance bioRxiv preprint doi: https://doi.org/10.1101/338368; this version posted June 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Proteomics reveals ablation of placental growth factor inhibits the insulin resistance 2 pathways in diabetic mouse retina 3 Madhu Sudhana Saddala1#, Anton Lennikov1# , Shibo Tang2,3, Hu Huang1,2,3* 4 5 1 Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States of America 6 2 Aier School of Ophthalmology, Central South University, Changsha, Hunan, China 7 3 Aier Eye Institute, Changsha, Hunan, China 8 9 *Corresponding author: 10 Hu Huang, PhD 11 Wilmer Eye Institute, Ophthalmology-Retinal Vascular Service 12 400 North Broadway St. Baltimore, MD, 21287 13 Hospital M017 Smith Building 14 Tel +1 (410)5020807 15 [email protected] 16 17 # Madhu Sudhana Saddala and Anton Lennikov have contributed equally to this work. 18 19 ORCID 20 Author 1: Madhu Sudhana Saddala ORCID: 0000-0002-6373-7080 21 Author 2: Anton Lennikov ORCID: 0000-0001-8625-1211 22 Author 3: Shibo Tang ORCID: 0000-0003-2737-6780 23 Author 4: Hu Huang ORCID: 0000-0003-2843-0320 24 1 bioRxiv preprint doi: https://doi.org/10.1101/338368; this version posted June 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 25 Acknowledgments 26 The authors wish to acknowledge the contribution of: Lijuan Fan for technical assistance, Dmitry Rumyancev for 27 artwork design, and Jianjiang Hao for MS analysis. 28 Funding: 29 This work was supported by NIH grant (EY027824) 30 Authors Contributions: 31 The study was conceived and designed by M.S.S., A.L. and H.H. H.H. performed the animal handling, sample 32 collection and in vivo examinations. The manuscript was written by M.S.S, A.L.,H.H. and critically revised by H.H. 33 and S.T. All Authors reviewed and accepted the final version of the manuscript. 34 Additional Information 35 Financial interest statement: 36 The authors have no financial interests to disclose in relation to this paper. 37 38 Conflict of interest statement: 39 The authors have no conflict of interests to disclose in relation to this paper. 40 2 bioRxiv preprint doi: https://doi.org/10.1101/338368; this version posted June 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 41 Abstract 42 The underlying molecular mechanisms that placental growth factor (PlGF) mediates the early complications at non- 43 proliferative diabetic retinopathy (DR) remain largely elusive. The objective of this study is to characterize 44 expression profile due to PlGF ablation in the retina of diabetic mice. The quantitative label-free proteomics was 45 carried out on retinal tissues collected from mouse strains (Akita; PlGF-/- and Akita.PlGF-/-). We have identified 46 3176 total proteins, and 107 were significantly different between the experimental groups, followed by gene 47 ontology, functional pathways, and protein-protein network interaction analysis. Gnb1, Gnb2, Gnb4, Gnai2, Gnao1, 48 Snap25, Stxbp1, Vamp2 and Gngt1 proteins are involved in insulin resistance pathways, which are down-regulated 49 in PlGF ablation in Akita diabetics (Akita.PlGF-/- vs. Akita), up-regulation in Akita vs. C57, PlGF-/- vs. C57. Prdx6, 50 Prdx5 (up-regulation) are known of antioxidant activity; Map2 is involved in neural protection pathways which are 51 up-regulated in Akita.PlGF-/- vs. Akita. Our results suggest that inhibition of insulin resistance pathway and the 52 enhancement of antioxidant defence and neural function may represent the potential mechanisms of anti-PlGF 53 compounds in the treatment of DR. 54 Total words: 175 55 56 57 58 59 Keywords: Diabetes, Akita mice, PlGF-knockout, Mass spectrometry, proteomics 60 3 bioRxiv preprint doi: https://doi.org/10.1101/338368; this version posted June 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 61 1.0 Introduction 62 Diabetic retinopathy (DR), a sight-threatening microvascular complication of diabetes myelitis (DM), remains the 63 leading cause of vision loss worldwide in the adult population, especially in economically developed countries. (Lee 64 et al, 2015) With the increasing number of people with DM, the prevalence of DR and diabetic macular edema 65 (DME) is expected to grow. (Wild et al, 2004) Metabolic changes in the diabetic retina result in the altered 66 expression pattern of some mediators including growth factors, neurotrophic factors, cytokines/chemokines, 67 vasoactive agents, and inflammatory and adhesion molecules, resulting in vascular lesions and cell death. (Chen et 68 al, 2015; Kowluru & Mishra, 2015; Liu et al, 2015) Emerging evidence suggests that retinal neurodegeneration is an 69 early event in the pathogenesis of DR which could participate in the development of microvascular abnormalities. 70 (Barber, 2015; Simo et al, 2014) Placenta growth factor (PlGF), a member of VEGF family proteins, first discovered 71 in human placental cDNA in 1991. 72 In over two decades of scientific research and development have increased our understanding of the PlGF biological 73 function. Despite the high level of expression in placenta, the ablation of PlGF in mice did not compromise the 74 healthy embryonic development or adverse postnatal health effects. (Carmeliet & Jain, 2011) Delivery of 75 recombinant PlGF homodimer, PlGF-VEGFA heterodimer significantly promoted angiogenesis in ischemic 76 conditions through FLT1. (Luttun et al, 2002) Furthermore, many other cell types express PlGF in pathological 77 conditions, including retinal pigment epithelial cells (RPE). (Hollborn et al, 2006) This upregulation is due not 78 only to hypoxia but also from stimulus including nitric oxide (Mohammed et al, 2007), cytokines, as interleukin 79 1 (IL-1) and TNF-α (De Ceuninck et al, 2004), and transforming growth factor-β1 (TGF-β). (Yao et al, 2005) 80 The observation further confirmed the specific role of PlGF in pathological conditions that during pathological 81 angiogenesis endothelial cells over-express PlGF. (Ponticelli et al, 2008) 82 Recently our group has reported PlGF deletion in C57BL/6-Ins2Akita/J (Akita) mouse line, containing a dominant 83 mutation that induces spontaneous diabetes with a rapid onset. (Barber et al, 2005) Ablation of PlGF in the diabetic 84 mice resulted in an decreased expression of diabetes-activated hypoxia-inducible factor (HIF)1α, vascular 85 endothelial growth factor (VEGF) pathway, including expression of HIF1α, VEGF, VEGFR1–3, and the extent of 86 phospho (p)-VEGFR1, p-VEGFR2, and p–endothelial nitric oxide synthase, in the retinas of diabetic PlGF−/− mice. 87 Without a noticeable effect on glucose balance or expression of intercellular adhesion molecule-1, vascular cell 88 adhesion molecule-1, CD11b, and CD18 (Huang et al, 2015a). 89 While many functions and biological roles of PlGF are still currently unknown, the transition to human patients with 90 two phase II clinical trials of anti-PlGF recombinant monoclonal antibody in human DR patients is currently 91 underway. (NCT03071068; NCT03499223; ThromboGenics, 2018) Use of PlGF antibodies in humans presents a 4 bioRxiv preprint doi: https://doi.org/10.1101/338368; this version posted June 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 92 challenge of better understanding the functions and pathways involved in PlGF knock out on the proteome scale. 93 Label-free mass spectrometry (LFMS) is a widely used tool for protein identification and quantification it is a gel- 94 free method allowing to conduct whole proteome analysis without the use of isotopic labeling (Luber et al, 2010). 95 Furthermore, as DR affects the expression of many commonly used “housekeeping” proteins such as ACTB and 96 Tubulin, MS approach resolve this issue as proteins are identified by the number of peptide sequences rather than 97 any relative quantification that requires a “housekeeping” protein (Rocha-Martins et al, 2012), (Li & Shen, 2013). In 98 the current study, we used label-free quantitative proteomics analysis to study retinal protein extracts from three 99 genetically modified mouse strains with diabetic and PlGF knockout condition as well as a combination of both to 100 further elucidate the molecular mechanisms of PlGF knockout playing beneficial role in DR on the proteome wide 101 level. We have identified 3176 total proteins, and 107 were significantly different between the experimental groups 102 (p<0.05). 103 104 1.0 Results 105 2.1 Animals and diabetic conditions. 106 Four 5-6 months old female mice were selected from each strain: C57BL/6-Ins2<Akita>/J.PlGF-/- (Akita.PlGF-/-), 107 PlGF-/-, C57BL/6-Ins2<Akita>/J (Akita), and C57BL/6J (C57) for this study. Graphical abstract of the animal 108 breeding program is presented in Figure 1A. Animals blood glucose, levels of glycated hemoglobin (HbA1c) and 109 body weight are presented in Table 1. All Akita mice have demonstrated significant increase of blood glucose (BG) 110 levels (p<0.001), HbA1c (p<0.05) and a decrease in body weight (p<0.01) when compared with C57 control animals 111 of the same age. Lack of PlGF did not affect blood glucose (p>0.05) levels of glycated hemoglobin (HbA1c) 112 (p>0.05) and body weight (p>0.05) for Akita.PlGF-/- vs. Akita. There was no significant difference in any of the 113 parameters in PlGF-/- vs. C57 mice of the same age. Retinal protein extracts 4 samples per group were trypsin 114 digested and subjected to the LC/MS/MS analysis (Figure 1C,D).
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